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Appendix
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Supplementary Table 1Strains, plasmids, and primers used in this study
Description
Reference
E. coli strains
JM109
recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1Δ(lacproAB)/F′[traD36 proAB+lacIq lacZΔM15]
Invitrogen
JM109 K8-11
JM109 derived, harboring φ80 attP inserted into
lacZ, yhiS, xylA, and malE sites
This study
EC100Dpir116
ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697
galUgalKλ-rpsLnupGpir-116(DHFR)
TransforMaxTM
T2
multiple copies of Ppdc-yfjBintegrated into JM109
This study
Plasmids
pAH57
pSC101 replicon, Xisλ and Intλ, AmpR
(Haldimann
and Wanner
2001)
pAH123
pSC101 replicon, Intφ80, AmpR
(Haldimann
and Wanner
2001)
pattP80
R6Kγreplicon, φ80attP, KanR
This study
pE76
Ppdcpromotercontrolled yfjB gene, AmpR
(Li et al. 2009)
p80T12
Two copies of Ppdc-yfjB inserted into pattP80, KanR
This study
pBHR68-gfp
gfp inserted into pBHR68, AmpR
This study
Primers
T1F
5’-GAAACACGGAGTGTCTAGATTACGCTCATGATCGC
T1R
5’-GAAAAAGCTTATTGCTAGCAGTAAAGAGGCTGG
T2F
5’-GAAAAAAGCTTTCTAGATTACGCTCATGATCGC
T2R
5’-GAAAAGCCACATAGGCATTGCTAGCAGTAAAGAGGCTG
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All oligonucleotides were synthesized by AuGCTBiotechnology (Beijing, China). Restriction
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endonuclease digestion sites were underlined.
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Supplementary Table 2qPCR primers used for transcriptional analysis
Primers
Sequence (5’-)
phaC_F
AAGTCCCAACCATTCAAG
phaC_R
AGAAGTCCTTCATGTAGC
phaA_F
ATCAAGAGCTATGCCAAC
phaA_R
GTTCACATTGACCTTGGA
phaB_F
GAGGTTGATGTGCTGATC
phaB_R
GACGAGATGTTGACGATG
yfjB_F
AAAGGTTACGAGGTCATC
yfjB_R
CGGTTGATTCCAATAACTT
pgi_F
TGTTCTTGGTAGCATCTAA
pgi_R
ATGTTGGCAGTATCAATAC
gapA_F
TACTACCGCTACTCAGAA
gapA_R
TCAGGTCAACTACAGATAC
zwf_F
AAGAGCAGCAATACAGAA
zwf_R
AGATTCAGTTCAGGTGTT
gnd_F
ACTGGTTCCTTACTATACG
gnd_R
TCACGATTACGACGAATA
icd_F
CTGAACGGTGACTACATT
icd_R
AGAATAATAGAGCCAGGATT
mdh_F
ATATCATTCGTTCCAACAC
mdh_R
CTTCAACCACTTCAGTAC
pntA_F
CGGAAGTGAAAGAACAAG
pntA_R
CGGTGGTGACAATGATAT
pntB_F
AAGTTGAAATGACCGAAAT
pntB_R
GATGAAGATACCGAGGAA
udhA_F
CGAAGAGTACGAGAAGAT
udhA_R
CTGATACATGCTGTTGAC
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Supplementary Table 3PCR primers used for genome integration verification
Primers
Integration site
K8_F
Sequence (5’-)
CAGTGAGCGCAACGCAATTA
lacZ
K8_R
GAAATACGGGCAGACATGGC
K9_F
TGGGGCGTGACATGAATATC
yhiS
K9_R
CAGGGACTTGTTCGCACCTT
K10_F
AGCGAGCGCACACTTGTGAA
xylA
K10_R
TATCGCTACCGATAACCGGG
K11_F
GGGAGGATGAGAACACGGCT
malE
K11_R
AGCACTGACCATTTCAGCGC
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Supplementary Fig.1PCR verification of NAD kinase integration
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PCR primers used for genome integration verification were listed in Supplementary Table 3.
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E. coli T2 was inoculated into Luria-Bertani medium and maintained by transferring into
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fresh medium every 12 h for 7 days. (A), original culture; (B), 7-day culture.
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Supplementary Fig.2Expression of gfp gene under the control of phaCAB promoter in E. coli
JM109 and T2
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E. coli JM109 and T2strains harboringpBHR68-gfp were cultivated in mineral saltsmedium at
37 °C for 48 h,respectively. Value 100 corresponds to equal gene expression relativeto
JM109 and values above 100 correspond to foldincrease. Data were the means of triplicate
experiments.
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